Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial. HSCs are rare and few can be found in thin tissue sections or upon live imaging, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as α-catulinGFP), we discover that α-catulinGFP is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of α-catulin−GFP+c-kit+ cells give long-term multilineage reconstitution of irradiated mice, indicating that α-catulin−GFP+c-kit+ cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of α-catulin–GFP+c-kit+ cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin–GFP+c-kit+ cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr+) and Cxcl12high niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67+) and non-dividing (Ki-67−), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr+Cxcl12high cells throughout the bone marrow.

[1]  Atsushi Miyawaki,et al.  Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain , 2011, Nature Neuroscience.

[2]  S. Rafii,et al.  Endothelial Jagged-1 is necessary for homeostatic and regenerative hematopoiesis. , 2013, Cell reports.

[3]  S. Rafii,et al.  Functional Heterogeneity of the Bone Marrow Vascular Niche , 2009, Annals of the New York Academy of Sciences.

[4]  S. Rafii,et al.  Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells , 2010, Nature Cell Biology.

[5]  T. Kume,et al.  Foxc1 is a critical regulator of haematopoietic stem/progenitor cell niche formation , 2014, Nature.

[6]  Xiaoqin Zhu,et al.  Age-dependent fate and lineage restriction of single NG2 cells , 2011, Development.

[7]  Shankar Srinivas,et al.  Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus , 2001, BMC Developmental Biology.

[8]  Qingming Luo,et al.  Recent progress in tissue optical clearing , 2013, Laser & photonics reviews.

[9]  S. Morrison,et al.  SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. , 2013, Cell stem cell.

[10]  Ben D. MacArthur,et al.  Mesenchymal and haematopoietic stem cells form a unique bone marrow niche , 2010, Nature.

[11]  Rajan P Kulkarni,et al.  Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing , 2014, Cell.

[12]  Frank Bradke,et al.  Three-dimensional imaging of solvent-cleared organs using 3DISCO , 2012, Nature Protocols.

[13]  D. Steindler,et al.  Neural stem and progenitor cells in nestin‐GFP transgenic mice , 2004, The Journal of comparative neurology.

[14]  Allan R. Jones,et al.  A robust and high-throughput Cre reporting and characterization system for the whole mouse brain , 2009, Nature Neuroscience.

[15]  S. Morrison,et al.  Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. , 2014, Cell stem cell.

[16]  G. Smyth,et al.  ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. , 2009, Journal of immunological methods.

[17]  N. Copeland,et al.  A highly efficient recombineering-based method for generating conditional knockout mutations. , 2003, Genome research.

[18]  David W. Rowe,et al.  Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche , 2009, Nature.

[19]  R. Weiler,et al.  Chemical Clearing and Dehydration of GFP Expressing Mouse Brains , 2012, PloS one.

[20]  B. Sacchetti,et al.  Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can Organize a Hematopoietic Microenvironment , 2007, Cell.

[21]  Elaine Dzierzak,et al.  Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos , 2012, Nature Protocols.

[22]  Reynaldo Sequerra,et al.  High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP , 2000, Nature Genetics.

[23]  A. Protopopov,et al.  Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment , 2013, Nature Cell Biology.

[24]  E. Susaki,et al.  Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis , 2014, Cell.

[25]  A. Schierloh,et al.  Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain , 2007, Nature Methods.

[26]  S. Morrison,et al.  The bone marrow niche for haematopoietic stem cells , 2014, Nature.

[27]  A. Miyawaki,et al.  Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow , 2009, The Journal of experimental medicine.

[28]  Y. Draenert,et al.  The vascular system of bone marrow. , 1980, Scanning electron microscopy.

[29]  Aaron S. Andalman,et al.  Structural and molecular interrogation of intact biological systems , 2013, Nature.

[30]  H. Dodt,et al.  Immunostaining, dehydration, and clearing of mouse embryos for ultramicroscopy. , 2013, Cold Spring Harbor protocols.

[31]  Zev Rosenwaks,et al.  Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. , 2009, Cell stem cell.

[32]  A. Iwama,et al.  Nonmyelinating Schwann Cells Maintain Hematopoietic Stem Cell Hibernation in the Bone Marrow Niche , 2011, Cell.

[33]  S. Morrison,et al.  Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance. , 2007, Cell stem cell.

[34]  N. Fujii,et al.  The essential functions of adipo-osteogenic progenitors as the hematopoietic stem and progenitor cell niche. , 2010, Immunity.

[35]  A. Bergman,et al.  Arteriolar niches maintain haematopoietic stem cell quiescence , 2013, Nature.

[36]  Lei Ding,et al.  Endothelial and perivascular cells maintain haematopoietic stem cells , 2011, Nature.

[37]  S. Morrison,et al.  Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches , 2013, Nature.

[38]  D. G. Osmond,et al.  Basement membrane of mouse bone marrow sinusoids shows distinctive structure and proteoglycan composition: A high resolution ultrastructural study , 2001, The Anatomical record.

[39]  S. Morrison,et al.  Supplemental Experimental Procedures , 2022 .

[40]  T. Nagasawa,et al.  Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. , 2006, Immunity.

[41]  F. van Roy,et al.  Human alpha-catulin, a novel alpha-catenin-like molecule with conserved genomic structure, but deviating alternative splicing. , 1999, Biochimica et biophysica acta.

[42]  Sergei A. Vinogradov,et al.  Direct measurement of local oxygen concentration in the bone marrow of live animals , 2014, Nature.

[43]  S. Morrison,et al.  Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1 , 2015, eLife.

[44]  K. Parmar,et al.  Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia , 2007, Proceedings of the National Academy of Sciences.

[45]  W. Slayton,et al.  High Levels of Acetylated Low-Density Lipoprotein Uptake and Low Tyrosine Kinase With Immunoglobulin and Epidermal Growth Factor Homology Domains-2 (Tie2) Promoter Activity Distinguish Sinusoids From Other Vessel Types in Murine Bone Marrow , 2009, Circulation.